Selenopeptides DOI: 10.1002/ange.201006939 Antioxidative Glutathione Peroxidase Activity of Selenoglutathione** Sari Yoshida, Fumio Kumakura, Itsuki Komatsu, Kenta Arai, Yuko Onuma, Hironobu Hojo,* Beena G. Singh, K. Indira Priyadarsini, and Michio Iwaoka* Selenoglutathione (GSeH), [1] a tripeptide comprising l-g- glutamic acid (Glu), l-selenocysteine (Sec), and glycine (Gly), is a selenium analogue of glutathione (GSH), a biologically important redox substrate. This unique seleno- peptide is attracting increasing attention as it was recently demonstrated that the oxidized form, that is, GSeSeG (1), is useful for oxidative folding of disulfide (SS)-containing pro- teins, such as bovine pancreatic ribonuclease A (RNase A) and trypsin inhibitor (BPTI). [2] Interestingly, diselenide 1 can even catalyze the folding reactions. [3] Due to the structural similarity to oxidized glutathione (GSSG), it is also a possible substrate for glutathione reductase (GR), [2] an enzyme that catalyzes reduction of GSSG with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. Thus, GSeH and GSeSeG (1) are promising targets to explore their unique biological functions. We recently succeeded in synthesizing a Sec derivative, N-(9-fluorenylmethoxycar- bonyl)-Se-(p-methoxyphenylmethyl)-l-selenocysteine (Fmoc-Sec(MPM)-OH, 2) using l-cystine as a starting material. [4] As an extension of our research, various Sec- containing peptides have now been synthesized from 2 by application of solid-phase peptide synthesis (SPPS), and their catalytic activity as an antioxidant has been investigated by employing the enzymatic assay of glutathione peroxidase (GPx), a well-known antioxidant selenoenzyme having a Sec residue at the redox-active site. [5] The Sec-containing peptides [6] targeted in this study involve 1 and oxidized dimers of tri- and pentapeptides (3 and 4, respectively) having an amino acid sequence of the GPx active site. These peptides were synthesized by SPPS using Fmoc-Gly Alko-PEG resin or Fmoc-Gly or Fmoc- Thr(tBu) Wang resin as a polymer support, by following the normal Fmoc methodology. The synthesized peptides were cleaved from the polymer support by using Reagent K [7] and reacted with trifluoroacetic acid (TFA) and 2,2’-dithiobis(5- nitropyridine) (DTNP) [8] to promote deprotection of the MPM group. The crude products were purified by reversed- phase (RP) HPLC using a Mightysil RP-18 column with a solvent gradient of acetonitrile (10–25 % in H 2 O), and fully characterized by MALDI-TOF mass spectrometry (MS) and amino acid analysis. GPx-like antioxidant activity of the synthesized peptides was first assayed according to the reaction shown in Scheme 1. [9] In this assay, the reduction rate of hydrogen peroxide (H 2 O 2 ) was monitored by a decrease in the UV absorbance at 340 nm resulting from NADPH, which was added to the assay solution to reduce GSSG (a counter- product of the H 2 O 2 reduction) to GSH in the presence of GR. Diselenides would enter the catalytic cycle either through oxidation to the corresponding selenenic acid (RSeOH) and seleninic acid (RSeO 2 H), or by reduction to the selenol (RSeH). A recent investigation on the GPx cycle of selenocystine ([H 2 N-Sec-OH] 2 , 5) revealed that the oxida- tion path is more feasible than the reduction path. [10] Initial velocities for the reduction of H 2 O 2 were measured in the presence of a catalytic amount of various diselenide compounds. The results are summarized in Table 1. When GSeSeG (1) was applied as a catalyst, the initial velocity for the reduction of H 2 O 2 (> 210 mm min À1 ) was largest among the tested diselenides including diphenyl diselenide (PhSeSePh), a commonly used standard material for the GPx assay, [11] thus indicating the prominent antioxidant activity. It is also notable that the catalytic activity decreases in the order 5 > 3 > 4, as amino acids are added to both sides of the Sec residue. The trend suggests that the local amino acid sequence at the GPx active site would not be essential for enhancement of the antioxidant activity. [12] The high GPx activity of 1 can be ascribed to several causes. One possible reason is that 1 participates in the reaction cycle shown in Scheme 2 because 1 is also a possible substrate for GR. [2] The presence of this bypass cycle was indeed confirmed by the distinct GPx activity observed in the Scheme 1. Catalytic cycle of the GSH-coupled GPx activity assay for a selenium catalyst. [*] S. Yoshida, F. Kumakura, I. Komatsu, K. Arai, Prof. Dr. M. Iwaoka Department of Chemistry, School of Science, Tokai University Kitakaname, Hiratsuka-shi, Kanagawa 259-1292 (Japan) Fax: (+ 81) 463-50-2094 E-mail: miwaoka@tokai.ac.jp Y. Onuma, Prof.Dr. H. Hojo Department of Applied Biochemistry, Tokai University Kitakaname, Hiratsuka-shi, Kanagawa 259-1292 (Japan) Dr. B. G. Singh, Dr. K. I. Priyadarsini Radiation and Photochemistry Division Bhabha Atomic Research Centre Trombay, Mumbai 400085 (India) [**] This work was supported by a Grant-in-Aid for Scientific Research (B) (No. 16350092) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the India–Japan Collaborative Science Programme (IJCSP; No. 08039221-000181). Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201006939. Angewandte Chemie 2173 Angew. Chem. 2011, 123, 2173 –2176  2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim